The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. This year we added the use of 3 dimensional electron microscopy and super optical resolution imaging to the technologies in the lab through strategic collaborations. The following major findings were made over the last year: 1) We completed a significant study in characterizing the structure of mitochondria in skeletal muscle and cardiac cells of the mouse using 3D electron microscopy, histochemical and super resolution microscopy. These studies revealed the presence of a mitochondrial reticulum that we proposed distributes energy rapidly throughout the cell via the mitochondrial membrane potential rather than the slow diffusion of molecules. The reticulum of the skeletal muscle and heart were similar in that the mitochondria are coupled via their membrane potential over large number of mitochondria in discrete regions of the cell. The skeletal muscle coupling occurred via both mitochondrial contact sites as well as long tubular structures running many microns across the short axis of the cell. In heart, the primary coupling mechanism was through mitochondrial contact sites along the long axis of the cell. In both systems we were able to demonstrate that there is a rapid conduction of the mitochondrial membrane potential across large regions of the cell. This electrical coupling, much like the wiring in a house, permits the rapid distribution of this primary potential energy for ATP production across the cell and summation of the energy from many mitochondria to specific regions in the cell. This tight coupling of mitochondria across the cell is also a risk. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have found that a rapid fail safe system is in place that rapidly removed damaged mitochondria from the network. Our current hypothesis is that this fail safe, or circuit breaker, mechanism is structural in nature representing the physical uncoupling of the mitochondria from the network. This rapid distribution of energy within the muscle cell contrasts with earlier models relying on slow high energy metabolite diffusion and provides another parameter to evaluate in different clinical conditions. We have also modeled how the electrical conduction occurs across the mitochondrial reticulum and reached the conclusion that the dominate ions, such as potassium, sodium and chloride must carry this current with appropriate proton-ion exchange systems. The role of the mitochondrial reticulum in different disease states associated with cardiac metabolic inadequacies, such as heart failure or ischemia, is yet to be investigated.